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Víctor Cifuentes - One of the best experts on this subject based on the ideXlab platform.
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sterol regulatory element binding protein sre1 promotes the synthesis of carotenoids and sterols in Xanthophyllomyces Dendrorhous
Frontiers in Microbiology, 2019Co-Authors: María Soledad Gutiérrez, Dionisia Sepulveda, Maria Fernandezlobato, Marcelo Baeza, Salvador Barahona, Ana-maría Gonzalez, Sebastian Campusano, Melissa Gomez, Peter J Espenshade, Víctor CifuentesAbstract:Xanthophyllomyces Dendrorhous is a basidiomycete yeast that synthesizes carotenoids, mainly astaxanthin, which are of great commercial interest. Currently, there are many unknown aspects related to regulatory mechanisms on the synthesis of carotenoids in this yeast. Our recent studies showed that changes in sterol levels and composition resulted in upregulation of genes in the mevalonate pathway required for the synthesis of carotenoid precursors, leading to increased production of these pigments. Sterol Regulatory Element-Binding Proteins (SREBP), called Sre1 in yeast, are conserved transcriptional regulators of sterol homeostasis and other cellular processes. Given the results linking sterols and carotenoids, we investigated the role of SREBP in sterol and carotenoid synthesis in X. Dendrorhous. In this study, we present the identification and functional characterization of the X. Dendrorhous SRE1 gene, which encodes the transcription factor Sre1. The deduced protein has the characteristic features of SREBP/Sre1 and binds to consensus DNA sequences in vitro. RNA-seq analysis and chromatin-immunoprecipitation experiments showed that genes of the mevalonate pathway and ergosterol biosynthesis are directly regulated by Sre1. The sre1- mutation reduced sterol and carotenoid production in X. Dendrorhous, and expression of the Sre1 N-terminal domain (Sre1N) increased carotenoid production more than twofold compared to wild-type. Overall, our results indicate that in X. Dendrorhous transcriptional regulation of genes in the mevalonate pathway control production of the isoprenoid derivatives, carotenoids and sterol. Our results provide new insights into the conserved regulatory functions of SREBP/Sre1 and identify pointing to the SREBP pathway as a potential target to enhance carotenoid production in X. Dendrorhous.
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Characterization of the cytochrome P450 monooxygenase genes (P450ome) from the carotenogenic yeast Xanthophyllomyces Dendrorhous
BMC Genomics, 2017Co-Authors: Pamela Córdova, Víctor Cifuentes, Marcelo Baeza, María Soledad Gutiérrez, Ana-maría Gonzalez, David R. Nelson, Jennifer AlcaínoAbstract:Background The cytochromes P450 (P450s) are a large superfamily of heme-containing monooxygenases involved in the oxidative metabolism of an enormous diversity of substrates. These enzymes require electrons for their activity, and the electrons are supplied by NAD(P)H through a P450 electron donor system, which is generally a cytochrome P450 reductase (CPR). The yeast Xanthophyllomyces Dendrorhous has evolved an exclusive P450-CPR system that specializes in the synthesis of astaxanthin, a carotenoid with commercial potential. For this reason, the aim of this work was to identify and characterize other potential P450 genes in the genome of this yeast using a bioinformatic approach. Results Thirteen potential P450-encoding genes were identified, and the analysis of their deduced proteins allowed them to be classified in ten different families: CYP51, CYP61, CYP5139 (with three members), CYP549A, CYP5491, CYP5492 (with two members), CYP5493, CYP53, CYP5494 and CYP5495. Structural analyses of the X. Dendrorhous P450 proteins showed that all of them have a predicted transmembrane region at their N-terminus and have the conserved domains characteristic of the P450s, including the heme-binding region (FxxGxRxCxG); the PER domain, with the characteristic signature for fungi (PxRW); the ExxR motif in the K-helix region and the oxygen-binding domain (OBD) (AGxDTT); also, the characteristic secondary structure elements of all the P450 proteins were identified. The possible functions of these P450s include primary, secondary and xenobiotic metabolism reactions such as sterol biosynthesis, carotenoid synthesis and aromatic compound degradation. Conclusions The carotenogenic yeast X. Dendrorhous has thirteen P450-encoding genes having potential functions in primary, secondary and xenobiotic metabolism reactions, including some genes of great interest for fatty acid hydroxylation and aromatic compound degradation. These findings established a basis for future studies about the role of P450s in the carotenogenic yeast X. Dendrorhous and their potential biotechnological applications.
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ral ssBioMed CentVirology Journal Open AcceResearch Polymorphism of viral dsRNA in Xanthophyllomyces Dendrorhous strains isolated from different geographic areas
2016Co-Authors: Marcelo Baeza, Mario Sanhueza, Oriana Flores, Vicente Oviedo, Diego Libkind, Víctor CifuentesAbstract:Background: Strains of the astaxanthin producing yeast Xanthophyllomyces Dendrorhous have been isolated from different cold regions around the earth, and the presence of double stranded RNA (dsRNA) elements was described in some isolates. This kind of viruses is widely distributed among yeasts and filamentous fungi and, although generally are cryptic in function, their studies have been a key factor in the knowledge of important fungi. In this work, the characterization and genetic relationships among dsRNA elements were determined in strains representatives of almost all regions of the earth where X. Dendrorhous have been isolated. Results: Almost all strains of X. Dendrorhous analyzed carry one, two or four dsRNA elements, of molecular sizes in the range from 0.8 to 5.0 kb. Different dsRNA-patterns were observed in strains with different geographic origin, being L1 (5.0 kb) the common dsRNA element. By hybridization assays a high genomic polymorphism was observed among L1 dsRNAs of different X. Dendrorhous strains. Contrary, hybridization was observed between L1 and L2 dsRNAs of strains from same or different regions, while the dsRNA elements of minor sizes (M, S1, and S2) present in several strains did not show hybridization with neither L1 or L2 dsRNAs. Along the growth curve of UCD 67-38
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molecular characterization and functional analysis of cytochrome b5 reductase cbr encoding genes from the carotenogenic yeast Xanthophyllomyces Dendrorhous
PLOS ONE, 2015Co-Authors: María Soledad Gutiérrez, Dionisia Sepulveda, Maria Cecilia Rojas, Víctor Cifuentes, Marcelo Baeza, Jennifer AlcaínoAbstract:The eukaryotic microsomal cytochrome P450 systems consist of a cytochrome P450 enzyme (P450) and a cytochrome P450 redox partner, which generally is a cytochrome P450 reductase (CPR) that supplies electrons from NADPH. However, alternative electron donors may exist such as cytochrome b5 reductase and cytochrome b5 (CBR and CYB5, respectively) via, which is NADH-dependent and are also anchored to the endoplasmic reticulum. In the carotenogenic yeast Xanthophyllomyces Dendrorhous, three P450-encoding genes have been described: crtS is involved in carotenogenesis and the CYP51 and CYP61 genes are both implicated in ergosterol biosynthesis. This yeast has a single CPR (encoded by the crtR gene), and a crtR- mutant does not produce astaxanthin. Considering that this mutant is viable, the existence of alternative cytochrome P450 electron donors like CBR and CYB5 could operate in this yeast. The aim of this work was to characterize the X. Dendrorhous CBR encoding gene and to study its involvement in P450 reactions in ergosterol and carotenoid biosynthesis. Two CBRs genes were identified (CBR.1 and CBR.2), and deletion mutants were constructed. The two mutants and the wild-type strain showed similar sterol production, with ergosterol being the main sterol produced. The crtR- mutant strain produced a lower proportion of ergosterol than did the parental strain. These results indicate that even though one of the two CBR genes could be involved in ergosterol biosynthesis, crtR complements their absence in the cbr- mutant strains, at least for ergosterol production. The higher NADH-dependent cytochrome c reductase activity together with the higher transcript levels of CBR.1 and CYB5 in the crtR- mutant as well as the lower NADH-dependent activity in CBS-cbr.1- strongly suggest that CBR.1-CYB5 via participates as an alternative electron donor pathway for P450 enzymes involved in ergosterol biosynthesis in X. Dendrorhous.
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Codon usage and codon context bias in Xanthophyllomyces Dendrorhous
BMC Genomics, 2015Co-Authors: Marcelo Baeza, Dionisia Sepulveda, Jennifer Alcaíno, Salvador Barahona, Víctor CifuentesAbstract:Background Synonymous codons are used differentially in organisms from the three domains of life, a phenomenon referred to as codon usage bias. In addition, codon pair bias, particularly in the 3’ codon context, has also been described in several organisms and is associated with the accuracy and rate of translation. An improved understanding of both types of bias is important for the optimization of heterologous protein expression, particularly in biotechnologically important organisms, such as the yeast Xanthophyllomyces Dendrorhous, a promising bioresource for the carotenoid astaxanthin. Using genomic and transcriptomic data, the codon usage and codon context biases of X. Dendrorhous open reading frames (ORFs) were analyzed to determine their expression levels, GC% and sequence lengths. X. Dendrorhous totiviral ORFs were also included in these analyses.
Jennifer Alcaíno - One of the best experts on this subject based on the ideXlab platform.
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Characterization of the cytochrome P450 monooxygenase genes (P450ome) from the carotenogenic yeast Xanthophyllomyces Dendrorhous
BMC Genomics, 2017Co-Authors: Pamela Córdova, Víctor Cifuentes, Marcelo Baeza, María Soledad Gutiérrez, Ana-maría Gonzalez, David R. Nelson, Jennifer AlcaínoAbstract:Background The cytochromes P450 (P450s) are a large superfamily of heme-containing monooxygenases involved in the oxidative metabolism of an enormous diversity of substrates. These enzymes require electrons for their activity, and the electrons are supplied by NAD(P)H through a P450 electron donor system, which is generally a cytochrome P450 reductase (CPR). The yeast Xanthophyllomyces Dendrorhous has evolved an exclusive P450-CPR system that specializes in the synthesis of astaxanthin, a carotenoid with commercial potential. For this reason, the aim of this work was to identify and characterize other potential P450 genes in the genome of this yeast using a bioinformatic approach. Results Thirteen potential P450-encoding genes were identified, and the analysis of their deduced proteins allowed them to be classified in ten different families: CYP51, CYP61, CYP5139 (with three members), CYP549A, CYP5491, CYP5492 (with two members), CYP5493, CYP53, CYP5494 and CYP5495. Structural analyses of the X. Dendrorhous P450 proteins showed that all of them have a predicted transmembrane region at their N-terminus and have the conserved domains characteristic of the P450s, including the heme-binding region (FxxGxRxCxG); the PER domain, with the characteristic signature for fungi (PxRW); the ExxR motif in the K-helix region and the oxygen-binding domain (OBD) (AGxDTT); also, the characteristic secondary structure elements of all the P450 proteins were identified. The possible functions of these P450s include primary, secondary and xenobiotic metabolism reactions such as sterol biosynthesis, carotenoid synthesis and aromatic compound degradation. Conclusions The carotenogenic yeast X. Dendrorhous has thirteen P450-encoding genes having potential functions in primary, secondary and xenobiotic metabolism reactions, including some genes of great interest for fatty acid hydroxylation and aromatic compound degradation. These findings established a basis for future studies about the role of P450s in the carotenogenic yeast X. Dendrorhous and their potential biotechnological applications.
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molecular characterization and functional analysis of cytochrome b5 reductase cbr encoding genes from the carotenogenic yeast Xanthophyllomyces Dendrorhous
PLOS ONE, 2015Co-Authors: María Soledad Gutiérrez, Dionisia Sepulveda, Maria Cecilia Rojas, Víctor Cifuentes, Marcelo Baeza, Jennifer AlcaínoAbstract:The eukaryotic microsomal cytochrome P450 systems consist of a cytochrome P450 enzyme (P450) and a cytochrome P450 redox partner, which generally is a cytochrome P450 reductase (CPR) that supplies electrons from NADPH. However, alternative electron donors may exist such as cytochrome b5 reductase and cytochrome b5 (CBR and CYB5, respectively) via, which is NADH-dependent and are also anchored to the endoplasmic reticulum. In the carotenogenic yeast Xanthophyllomyces Dendrorhous, three P450-encoding genes have been described: crtS is involved in carotenogenesis and the CYP51 and CYP61 genes are both implicated in ergosterol biosynthesis. This yeast has a single CPR (encoded by the crtR gene), and a crtR- mutant does not produce astaxanthin. Considering that this mutant is viable, the existence of alternative cytochrome P450 electron donors like CBR and CYB5 could operate in this yeast. The aim of this work was to characterize the X. Dendrorhous CBR encoding gene and to study its involvement in P450 reactions in ergosterol and carotenoid biosynthesis. Two CBRs genes were identified (CBR.1 and CBR.2), and deletion mutants were constructed. The two mutants and the wild-type strain showed similar sterol production, with ergosterol being the main sterol produced. The crtR- mutant strain produced a lower proportion of ergosterol than did the parental strain. These results indicate that even though one of the two CBR genes could be involved in ergosterol biosynthesis, crtR complements their absence in the cbr- mutant strains, at least for ergosterol production. The higher NADH-dependent cytochrome c reductase activity together with the higher transcript levels of CBR.1 and CYB5 in the crtR- mutant as well as the lower NADH-dependent activity in CBS-cbr.1- strongly suggest that CBR.1-CYB5 via participates as an alternative electron donor pathway for P450 enzymes involved in ergosterol biosynthesis in X. Dendrorhous.
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Codon usage and codon context bias in Xanthophyllomyces Dendrorhous
BMC Genomics, 2015Co-Authors: Marcelo Baeza, Dionisia Sepulveda, Jennifer Alcaíno, Salvador Barahona, Víctor CifuentesAbstract:Background Synonymous codons are used differentially in organisms from the three domains of life, a phenomenon referred to as codon usage bias. In addition, codon pair bias, particularly in the 3’ codon context, has also been described in several organisms and is associated with the accuracy and rate of translation. An improved understanding of both types of bias is important for the optimization of heterologous protein expression, particularly in biotechnologically important organisms, such as the yeast Xanthophyllomyces Dendrorhous, a promising bioresource for the carotenoid astaxanthin. Using genomic and transcriptomic data, the codon usage and codon context biases of X. Dendrorhous open reading frames (ORFs) were analyzed to determine their expression levels, GC% and sequence lengths. X. Dendrorhous totiviral ORFs were also included in these analyses.
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functional characterization of the Xanthophyllomyces Dendrorhous farnesyl pyrophosphate synthase and geranylgeranyl pyrophosphate synthase encoding genes that are involved in the synthesis of isoprenoid precursors
PLOS ONE, 2014Co-Authors: Jennifer Alcaíno, Mauricio Niklitschek, Dionisia Sepulveda, Maria Cecilia Rojas, Ignacio Romero, Marcelo Baeza, Víctor CifuentesAbstract:The yeast Xanthophyllomyces Dendrorhous synthesizes the carotenoid astaxanthin, which has applications in biotechnology because of its antioxidant and pigmentation properties. However, wild-type strains produce too low amounts of carotenoids to be industrially competitive. Considering this background, it is indispensable to understand how the synthesis of astaxanthin is controlled and regulated in this yeast. In this work, the steps leading to the synthesis of the carotenoid precursor geranylgeranyl pyrophosphate (GGPP, C20) in X. Dendrorhous from isopentenyl pyrophosphate (IPP, C5) and dimethylallyl pyrophosphate (DMAPP, C5) was characterized. Two prenyl transferase encoding genes, FPS and crtE, were expressed in E. coli. The enzymatic assays using recombinant E. coli protein extracts demonstrated that FPS and crtE encode a farnesyl pyrophosphate (FPP, C15) synthase and a GGPP-synthase, respectively. X. Dendrorhous FPP-synthase produces geranyl pyrophosphate (GPP, C10) from IPP and DMAPP and FPP from IPP and GPP, while the X. Dendrorhous GGPP-synthase utilizes only FPP and IPP as substrates to produce GGPP. Additionally, the FPS and crtE genes were over-expressed in X. Dendrorhous, resulting in an increase of the total carotenoid production. Because the parental strain is diploid, the deletion of one of the alleles of these genes did not affect the total carotenoid production, but the composition was significantly altered. These results suggest that the over-expression of these genes might provoke a higher carbon flux towards carotenogenesis, most likely involving an earlier formation of a carotenogenic enzyme complex. Conversely, the lower carbon flux towards carotenogenesis in the deletion mutants might delay or lead to a partial formation of a carotenogenic enzyme complex, which could explain the accumulation of astaxanthin carotenoid precursors in these mutants. In conclusion, the FPS and the crtE genes represent good candidates to manipulate to favor carotenoid biosynthesis in X. Dendrorhous.
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Enhancement of carotenoid production by disrupting the C22-sterol desaturase gene (CYP61) in Xanthophyllomyces Dendrorhous
BMC Microbiology, 2012Co-Authors: Iris Loto, Dionisia Sepulveda, Víctor Cifuentes, Marcelo Baeza, Salvador Barahona, Pilar Martinez-moya, María Soledad Gutiérrez, Jennifer AlcaínoAbstract:Background Xanthophyllomyces Dendrorhous is a basidiomycetous yeast that synthesizes astaxanthin, which is a carotenoid with a great biotechnological impact. The ergosterol and carotenoid synthesis pathways are derived from the mevalonate pathway, and in both pathways, cytochrome P450 enzymes are involved. Results In this study, we isolated and described the X. Dendrorhous CYP61 gene, which encodes a cytochrome P450 involved in ergosterol biosynthesis. This gene is composed of nine exons and encodes a 526 amino acid polypeptide that shares significant percentages of identity and similitude with the C22-sterol desaturase, CYP61, from other fungi. Mutants derived from different parental strains were obtained by disrupting the CYP61 gene with an antibiotic selection marker. These mutants were not able to produce ergosterol and accumulated ergosta-5,8,22-trien-3-ol and ergosta-5,8-dien-3-ol. Interestingly, all of the mutants had a more intense red color phenotype than their respective parental strains. The carotenoid composition was qualitatively and quantitatively analyzed by RP-HPLC, revealing that the carotenoid content was higher in the mutant strains without major changes in their composition. The expression of the HMGR gene, which encodes an enzyme involved in the mevalonate pathway (3-hydroxy-3-methylglutaryl-CoA reductase), was analyzed by RT-qPCR showing that its transcript levels are higher in the CYP61 mutants. Conclusions These results suggest that in X. Dendrorhous , ergosterol regulates HMGR gene expression by a negative feedback mechanism and in this way; it contributes in the regulation of the carotenoid biosynthesis.
Marcelo Baeza - One of the best experts on this subject based on the ideXlab platform.
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sterol regulatory element binding protein sre1 promotes the synthesis of carotenoids and sterols in Xanthophyllomyces Dendrorhous
Frontiers in Microbiology, 2019Co-Authors: María Soledad Gutiérrez, Dionisia Sepulveda, Maria Fernandezlobato, Marcelo Baeza, Salvador Barahona, Ana-maría Gonzalez, Sebastian Campusano, Melissa Gomez, Peter J Espenshade, Víctor CifuentesAbstract:Xanthophyllomyces Dendrorhous is a basidiomycete yeast that synthesizes carotenoids, mainly astaxanthin, which are of great commercial interest. Currently, there are many unknown aspects related to regulatory mechanisms on the synthesis of carotenoids in this yeast. Our recent studies showed that changes in sterol levels and composition resulted in upregulation of genes in the mevalonate pathway required for the synthesis of carotenoid precursors, leading to increased production of these pigments. Sterol Regulatory Element-Binding Proteins (SREBP), called Sre1 in yeast, are conserved transcriptional regulators of sterol homeostasis and other cellular processes. Given the results linking sterols and carotenoids, we investigated the role of SREBP in sterol and carotenoid synthesis in X. Dendrorhous. In this study, we present the identification and functional characterization of the X. Dendrorhous SRE1 gene, which encodes the transcription factor Sre1. The deduced protein has the characteristic features of SREBP/Sre1 and binds to consensus DNA sequences in vitro. RNA-seq analysis and chromatin-immunoprecipitation experiments showed that genes of the mevalonate pathway and ergosterol biosynthesis are directly regulated by Sre1. The sre1- mutation reduced sterol and carotenoid production in X. Dendrorhous, and expression of the Sre1 N-terminal domain (Sre1N) increased carotenoid production more than twofold compared to wild-type. Overall, our results indicate that in X. Dendrorhous transcriptional regulation of genes in the mevalonate pathway control production of the isoprenoid derivatives, carotenoids and sterol. Our results provide new insights into the conserved regulatory functions of SREBP/Sre1 and identify pointing to the SREBP pathway as a potential target to enhance carotenoid production in X. Dendrorhous.
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Characterization of the cytochrome P450 monooxygenase genes (P450ome) from the carotenogenic yeast Xanthophyllomyces Dendrorhous
BMC Genomics, 2017Co-Authors: Pamela Córdova, Víctor Cifuentes, Marcelo Baeza, María Soledad Gutiérrez, Ana-maría Gonzalez, David R. Nelson, Jennifer AlcaínoAbstract:Background The cytochromes P450 (P450s) are a large superfamily of heme-containing monooxygenases involved in the oxidative metabolism of an enormous diversity of substrates. These enzymes require electrons for their activity, and the electrons are supplied by NAD(P)H through a P450 electron donor system, which is generally a cytochrome P450 reductase (CPR). The yeast Xanthophyllomyces Dendrorhous has evolved an exclusive P450-CPR system that specializes in the synthesis of astaxanthin, a carotenoid with commercial potential. For this reason, the aim of this work was to identify and characterize other potential P450 genes in the genome of this yeast using a bioinformatic approach. Results Thirteen potential P450-encoding genes were identified, and the analysis of their deduced proteins allowed them to be classified in ten different families: CYP51, CYP61, CYP5139 (with three members), CYP549A, CYP5491, CYP5492 (with two members), CYP5493, CYP53, CYP5494 and CYP5495. Structural analyses of the X. Dendrorhous P450 proteins showed that all of them have a predicted transmembrane region at their N-terminus and have the conserved domains characteristic of the P450s, including the heme-binding region (FxxGxRxCxG); the PER domain, with the characteristic signature for fungi (PxRW); the ExxR motif in the K-helix region and the oxygen-binding domain (OBD) (AGxDTT); also, the characteristic secondary structure elements of all the P450 proteins were identified. The possible functions of these P450s include primary, secondary and xenobiotic metabolism reactions such as sterol biosynthesis, carotenoid synthesis and aromatic compound degradation. Conclusions The carotenogenic yeast X. Dendrorhous has thirteen P450-encoding genes having potential functions in primary, secondary and xenobiotic metabolism reactions, including some genes of great interest for fatty acid hydroxylation and aromatic compound degradation. These findings established a basis for future studies about the role of P450s in the carotenogenic yeast X. Dendrorhous and their potential biotechnological applications.
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ral ssBioMed CentVirology Journal Open AcceResearch Polymorphism of viral dsRNA in Xanthophyllomyces Dendrorhous strains isolated from different geographic areas
2016Co-Authors: Marcelo Baeza, Mario Sanhueza, Oriana Flores, Vicente Oviedo, Diego Libkind, Víctor CifuentesAbstract:Background: Strains of the astaxanthin producing yeast Xanthophyllomyces Dendrorhous have been isolated from different cold regions around the earth, and the presence of double stranded RNA (dsRNA) elements was described in some isolates. This kind of viruses is widely distributed among yeasts and filamentous fungi and, although generally are cryptic in function, their studies have been a key factor in the knowledge of important fungi. In this work, the characterization and genetic relationships among dsRNA elements were determined in strains representatives of almost all regions of the earth where X. Dendrorhous have been isolated. Results: Almost all strains of X. Dendrorhous analyzed carry one, two or four dsRNA elements, of molecular sizes in the range from 0.8 to 5.0 kb. Different dsRNA-patterns were observed in strains with different geographic origin, being L1 (5.0 kb) the common dsRNA element. By hybridization assays a high genomic polymorphism was observed among L1 dsRNAs of different X. Dendrorhous strains. Contrary, hybridization was observed between L1 and L2 dsRNAs of strains from same or different regions, while the dsRNA elements of minor sizes (M, S1, and S2) present in several strains did not show hybridization with neither L1 or L2 dsRNAs. Along the growth curve of UCD 67-38
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RESEARCH ARTICLE Molecular Characterization and Functional Analysis of Cytochrome b5 Reductase (CBR) Encoding Genes from the Carotenogenic Yeast Xanthophyllomyces Dendrorhous
2016Co-Authors: María Soledad Gutiérrez, Dionisia Sepulveda, Maria Cecilia Rojas, Marcelo Baeza, Víctor Cifuentes JenniferAbstract:The eukaryotic microsomal cytochrome P450 systems consist of a cytochrome P450 enzyme (P450) and a cytochrome P450 redox partner, which generally is a cytochrome P450 reductase (CPR) that supplies electrons from NADPH. However, alternative electron donors may exist such as cytochrome b5 reductase and cytochrome b5 (CBR and CYB5, respectively) via, which is NADH-dependent and are also anchored to the endoplasmic reticulum. In the carotenogenic yeast Xanthophyllomyces Dendrorhous, three P450-encod-ing genes have been described: crtS is involved in carotenogenesis and the CYP51 and CYP61 genes are both implicated in ergosterol biosynthesis. This yeast has a single CPR (encoded by the crtR gene), and a crtR- mutant does not produce astaxanthin. Considering that this mutant is viable, the existence of alternative cytochrome P450 electron donors like CBR and CYB5 could operate in this yeast. The aim of this work was to characterize the X. Dendrorhous CBR encoding gene and to study its involvement in P450 reactions in ergos-terol and carotenoid biosynthesis. Two CBRs genes were identified (CBR.1 and CBR.2), and deletion mutants were constructed. The two mutants and the wild-type strain showe
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molecular characterization and functional analysis of cytochrome b5 reductase cbr encoding genes from the carotenogenic yeast Xanthophyllomyces Dendrorhous
PLOS ONE, 2015Co-Authors: María Soledad Gutiérrez, Dionisia Sepulveda, Maria Cecilia Rojas, Víctor Cifuentes, Marcelo Baeza, Jennifer AlcaínoAbstract:The eukaryotic microsomal cytochrome P450 systems consist of a cytochrome P450 enzyme (P450) and a cytochrome P450 redox partner, which generally is a cytochrome P450 reductase (CPR) that supplies electrons from NADPH. However, alternative electron donors may exist such as cytochrome b5 reductase and cytochrome b5 (CBR and CYB5, respectively) via, which is NADH-dependent and are also anchored to the endoplasmic reticulum. In the carotenogenic yeast Xanthophyllomyces Dendrorhous, three P450-encoding genes have been described: crtS is involved in carotenogenesis and the CYP51 and CYP61 genes are both implicated in ergosterol biosynthesis. This yeast has a single CPR (encoded by the crtR gene), and a crtR- mutant does not produce astaxanthin. Considering that this mutant is viable, the existence of alternative cytochrome P450 electron donors like CBR and CYB5 could operate in this yeast. The aim of this work was to characterize the X. Dendrorhous CBR encoding gene and to study its involvement in P450 reactions in ergosterol and carotenoid biosynthesis. Two CBRs genes were identified (CBR.1 and CBR.2), and deletion mutants were constructed. The two mutants and the wild-type strain showed similar sterol production, with ergosterol being the main sterol produced. The crtR- mutant strain produced a lower proportion of ergosterol than did the parental strain. These results indicate that even though one of the two CBR genes could be involved in ergosterol biosynthesis, crtR complements their absence in the cbr- mutant strains, at least for ergosterol production. The higher NADH-dependent cytochrome c reductase activity together with the higher transcript levels of CBR.1 and CYB5 in the crtR- mutant as well as the lower NADH-dependent activity in CBS-cbr.1- strongly suggest that CBR.1-CYB5 via participates as an alternative electron donor pathway for P450 enzymes involved in ergosterol biosynthesis in X. Dendrorhous.
Gerhard Sandmann - One of the best experts on this subject based on the ideXlab platform.
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combinatorial biosynthesis of novel multi hydroxy carotenoids in the red yeast Xanthophyllomyces Dendrorhous
Journal of Fungi, 2017Co-Authors: Hendrik Pollmann, Jürgen Breitenbach, Hendrik Wolff, Helge B Bode, Gerhard SandmannAbstract:The red yeast Xanthophyllomyces Dendrorhous is an established platform for the synthesis of carotenoids. It was used for the generation of novel multi oxygenated carotenoid structures. This was achieved by a combinatorial approach starting with the selection of a β-carotene accumulating mutant, stepwise pathway engineering by integration of three microbial genes into the genome and finally the chemical reduction of the resulting 4,4’-diketo-nostoxanthin (2,3,2’,3’-tetrahydroxy-4,4’-diketo-β-carotene) and 4-keto-nostoxanthin (2,3,2’,3’-tetrahydroxy-4-monoketo-β-carotene). Both keto carotenoids and the resulting 4,4’-dihydroxy-nostoxanthin (2,3,4,2’,3’,4’-hexahydroxy-β-carotene) and 4-hydroxy-nostoxanthin (2,3,4,2’3’-pentahydroxy-β-carotene) were separated by high-performance liquid chromatography (HPLC) and analyzed by mass spectrometry. Their molecular masses and fragmentation patterns allowed the unequivocal identification of all four carotenoids.
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The genome of the basal agaricomycete Xanthophyllomyces Dendrorhous provides insights into the organization of its acetyl-CoA derived pathways and the evolution of Agaricomycotina
BMC Genomics, 2015Co-Authors: Rahul Sharma, Sören Gassel, Sabine Steiger, Gerhard Sandmann, Robert Bauer, Marco ThinesAbstract:Background Xanthophyllomyces Dendrorhous is a basal agaricomycete with uncertain taxonomic placement, known for its unique ability to produce astaxanthin, a carotenoid with antioxidant properties. It was the aim of this study to elucidate the organization of its CoA-derived pathways and to use the genomic information of X. Dendrorhous for a phylogenomic investigation of the Basidiomycota. Results The genome assembly of a haploid strain of Xanthophyllomyces Dendrorhous revealed a genome of 19.50 Megabases with 6385 protein coding genes. Phylogenetic analyses were conducted including 48 fungal genomes. These revealed Ustilaginomycotina and Agaricomycotina as sister groups. In the latter a well-supported sister-group relationship of two major orders, Polyporales and Russulales, was inferred. Wallemia occupies a basal position within the Agaricomycotina and X. Dendrorhous represents the basal lineage of the Tremellomycetes, highlighting that the typical tremelloid parenthesomes have either convergently evolved in Wallemia and the Tremellomycetes, or were lost in the Cystofilobasidiales lineage. A detailed characterization of the CoA-related pathways was done and all genes for fatty acid, sterol and carotenoid synthesis have been assigned. Conclusions The current study ascertains that Wallemia with tremelloid parenthesomes is the most basal agaricomycotinous lineage and that Cystofilobasidiales without tremelloid parenthesomes are deeply rooted within Tremellomycetes, suggesting that parenthesomes at septal pores might be the core synapomorphy for the Agaricomycotina. Apart from evolutionary insights the genome sequence of X. Dendrorhous will facilitate genetic pathway engineering for optimized astaxanthin or oxidative alcohol production.
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The genome of the basal agaricomycete Xanthophyllomyces Dendrorhous provides insights into the organization of its acetyl-CoA derived pathways and the evolution of Agaricomycotina
BMC Genomics, 2015Co-Authors: Rahul Sharma, Sören Gassel, Sabine Steiger, Gerhard Sandmann, Robert Bauer, Marco ThinesAbstract:Background Xanthophyllomyces Dendrorhous is a basal agaricomycete with uncertain taxonomic placement, known for its unique ability to produce astaxanthin, a carotenoid with antioxidant properties. It was the aim of this study to elucidate the organization of its CoA-derived pathways and to use the genomic information of X. Dendrorhous for a phylogenomic investigation of the Basidiomycota.
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engineering of geranylgeranyl pyrophosphate synthase levels and physiological conditions for enhanced carotenoid and astaxanthin synthesis in Xanthophyllomyces Dendrorhous
Biotechnology Letters, 2011Co-Authors: Jürgen Breitenbach, Jan C Verdoes, Albert J J Van Ooyen, Hans De Visser, Gerhard SandmannAbstract:The basidiomycetous yeast, Xanthophyllomyces Dendrorhous, is one of the very few organisms which can be used for biological production of the carotenoid astaxanthin. crtE cDNA has been cloned from this fungus for engineering of the terpenoid pathway. The function of its gene product as a geranylgeranyl pyrophosphate synthase was established. X. Dendrorhous was transformed with the crtE cDNA to divert metabolite flow from the sterol pathway towards carotenoid biosynthesis. Transformants were obtained with increased levels of geranylgeranyl pyrophosphate synthase leading to higher carotenoid levels including astaxanthin. Physiological conditions for maximum carotenoid synthesis for wild type and the CrtE transformant were dim light and extra air supply of the shaking culture. These conditions and the transformation with crtE had additive effects and resulted in an 8-fold higher astaxanthin formation as compared to the initial wild type culture without illumination and extra air supply yielding 451 μg/g dry wt within 4 days of growth.
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high level production of beta carotene in saccharomyces cerevisiae by successive transformation with carotenogenic genes from Xanthophyllomyces Dendrorhous
Applied and Environmental Microbiology, 2007Co-Authors: Rene Verwaal, Gerhard Sandmann, Hans De Visser, Jing Wang, Jeanpaul Meijnen, Johan A Van Den Berg, Albert J J Van OoyenAbstract:To determine whether Saccharomyces cerevisiae can serve as a host for efficient carotenoid and especially β-carotene production, carotenogenic genes from the carotenoid-producing yeast Xanthophyllomyces Dendrorhous were introduced and overexpressed in S. cerevisiae. Because overexpression of these genes from an episomal expression vector resulted in unstable strains, the genes were integrated into genomic DNA to yield stable, carotenoid-producing S. cerevisiae cells. Furthermore, carotenoid production levels were higher in strains containing integrated carotenogenic genes. Overexpression of crtYB (which encodes a bifunctional phytoene synthase and lycopene cyclase) and crtI (phytoene desaturase) from X. Dendrorhous was sufficient to enable carotenoid production. Carotenoid production levels were increased by additional overexpression of a homologous geranylgeranyl diphosphate (GGPP) synthase from S. cerevisiae that is encoded by BTS1. Combined overexpression of crtE (heterologous GGPP synthase) from X. Dendrorhous with crtYB and crtI and introduction of an additional copy of a truncated 3-hydroxy-3-methylglutaryl-coenzyme A reductase gene (tHMG1) into carotenoid-producing cells resulted in a successive increase in carotenoid production levels. The strains mentioned produced high levels of intermediates of the carotenogenic pathway and comparable low levels of the preferred end product β-carotene, as determined by high-performance liquid chromatography. We finally succeeded in constructing an S. cerevisiae strain capable of producing high levels of β-carotene, up to 5.9 mg/g (dry weight), which was accomplished by the introduction of an additional copy of crtI and tHMG1 into carotenoid-producing yeast cells. This transformant is promising for further development toward the biotechnological production of β-carotene by S. cerevisiae.
Dionisia Sepulveda - One of the best experts on this subject based on the ideXlab platform.
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sterol regulatory element binding protein sre1 promotes the synthesis of carotenoids and sterols in Xanthophyllomyces Dendrorhous
Frontiers in Microbiology, 2019Co-Authors: María Soledad Gutiérrez, Dionisia Sepulveda, Maria Fernandezlobato, Marcelo Baeza, Salvador Barahona, Ana-maría Gonzalez, Sebastian Campusano, Melissa Gomez, Peter J Espenshade, Víctor CifuentesAbstract:Xanthophyllomyces Dendrorhous is a basidiomycete yeast that synthesizes carotenoids, mainly astaxanthin, which are of great commercial interest. Currently, there are many unknown aspects related to regulatory mechanisms on the synthesis of carotenoids in this yeast. Our recent studies showed that changes in sterol levels and composition resulted in upregulation of genes in the mevalonate pathway required for the synthesis of carotenoid precursors, leading to increased production of these pigments. Sterol Regulatory Element-Binding Proteins (SREBP), called Sre1 in yeast, are conserved transcriptional regulators of sterol homeostasis and other cellular processes. Given the results linking sterols and carotenoids, we investigated the role of SREBP in sterol and carotenoid synthesis in X. Dendrorhous. In this study, we present the identification and functional characterization of the X. Dendrorhous SRE1 gene, which encodes the transcription factor Sre1. The deduced protein has the characteristic features of SREBP/Sre1 and binds to consensus DNA sequences in vitro. RNA-seq analysis and chromatin-immunoprecipitation experiments showed that genes of the mevalonate pathway and ergosterol biosynthesis are directly regulated by Sre1. The sre1- mutation reduced sterol and carotenoid production in X. Dendrorhous, and expression of the Sre1 N-terminal domain (Sre1N) increased carotenoid production more than twofold compared to wild-type. Overall, our results indicate that in X. Dendrorhous transcriptional regulation of genes in the mevalonate pathway control production of the isoprenoid derivatives, carotenoids and sterol. Our results provide new insights into the conserved regulatory functions of SREBP/Sre1 and identify pointing to the SREBP pathway as a potential target to enhance carotenoid production in X. Dendrorhous.
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RESEARCH ARTICLE Molecular Characterization and Functional Analysis of Cytochrome b5 Reductase (CBR) Encoding Genes from the Carotenogenic Yeast Xanthophyllomyces Dendrorhous
2016Co-Authors: María Soledad Gutiérrez, Dionisia Sepulveda, Maria Cecilia Rojas, Marcelo Baeza, Víctor Cifuentes JenniferAbstract:The eukaryotic microsomal cytochrome P450 systems consist of a cytochrome P450 enzyme (P450) and a cytochrome P450 redox partner, which generally is a cytochrome P450 reductase (CPR) that supplies electrons from NADPH. However, alternative electron donors may exist such as cytochrome b5 reductase and cytochrome b5 (CBR and CYB5, respectively) via, which is NADH-dependent and are also anchored to the endoplasmic reticulum. In the carotenogenic yeast Xanthophyllomyces Dendrorhous, three P450-encod-ing genes have been described: crtS is involved in carotenogenesis and the CYP51 and CYP61 genes are both implicated in ergosterol biosynthesis. This yeast has a single CPR (encoded by the crtR gene), and a crtR- mutant does not produce astaxanthin. Considering that this mutant is viable, the existence of alternative cytochrome P450 electron donors like CBR and CYB5 could operate in this yeast. The aim of this work was to characterize the X. Dendrorhous CBR encoding gene and to study its involvement in P450 reactions in ergos-terol and carotenoid biosynthesis. Two CBRs genes were identified (CBR.1 and CBR.2), and deletion mutants were constructed. The two mutants and the wild-type strain showe
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molecular characterization and functional analysis of cytochrome b5 reductase cbr encoding genes from the carotenogenic yeast Xanthophyllomyces Dendrorhous
PLOS ONE, 2015Co-Authors: María Soledad Gutiérrez, Dionisia Sepulveda, Maria Cecilia Rojas, Víctor Cifuentes, Marcelo Baeza, Jennifer AlcaínoAbstract:The eukaryotic microsomal cytochrome P450 systems consist of a cytochrome P450 enzyme (P450) and a cytochrome P450 redox partner, which generally is a cytochrome P450 reductase (CPR) that supplies electrons from NADPH. However, alternative electron donors may exist such as cytochrome b5 reductase and cytochrome b5 (CBR and CYB5, respectively) via, which is NADH-dependent and are also anchored to the endoplasmic reticulum. In the carotenogenic yeast Xanthophyllomyces Dendrorhous, three P450-encoding genes have been described: crtS is involved in carotenogenesis and the CYP51 and CYP61 genes are both implicated in ergosterol biosynthesis. This yeast has a single CPR (encoded by the crtR gene), and a crtR- mutant does not produce astaxanthin. Considering that this mutant is viable, the existence of alternative cytochrome P450 electron donors like CBR and CYB5 could operate in this yeast. The aim of this work was to characterize the X. Dendrorhous CBR encoding gene and to study its involvement in P450 reactions in ergosterol and carotenoid biosynthesis. Two CBRs genes were identified (CBR.1 and CBR.2), and deletion mutants were constructed. The two mutants and the wild-type strain showed similar sterol production, with ergosterol being the main sterol produced. The crtR- mutant strain produced a lower proportion of ergosterol than did the parental strain. These results indicate that even though one of the two CBR genes could be involved in ergosterol biosynthesis, crtR complements their absence in the cbr- mutant strains, at least for ergosterol production. The higher NADH-dependent cytochrome c reductase activity together with the higher transcript levels of CBR.1 and CYB5 in the crtR- mutant as well as the lower NADH-dependent activity in CBS-cbr.1- strongly suggest that CBR.1-CYB5 via participates as an alternative electron donor pathway for P450 enzymes involved in ergosterol biosynthesis in X. Dendrorhous.
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Codon usage and codon context bias in Xanthophyllomyces Dendrorhous
BMC Genomics, 2015Co-Authors: Marcelo Baeza, Dionisia Sepulveda, Jennifer Alcaíno, Salvador Barahona, Víctor CifuentesAbstract:Background Synonymous codons are used differentially in organisms from the three domains of life, a phenomenon referred to as codon usage bias. In addition, codon pair bias, particularly in the 3’ codon context, has also been described in several organisms and is associated with the accuracy and rate of translation. An improved understanding of both types of bias is important for the optimization of heterologous protein expression, particularly in biotechnologically important organisms, such as the yeast Xanthophyllomyces Dendrorhous, a promising bioresource for the carotenoid astaxanthin. Using genomic and transcriptomic data, the codon usage and codon context biases of X. Dendrorhous open reading frames (ORFs) were analyzed to determine their expression levels, GC% and sequence lengths. X. Dendrorhous totiviral ORFs were also included in these analyses.
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functional characterization of the Xanthophyllomyces Dendrorhous farnesyl pyrophosphate synthase and geranylgeranyl pyrophosphate synthase encoding genes that are involved in the synthesis of isoprenoid precursors
PLOS ONE, 2014Co-Authors: Jennifer Alcaíno, Mauricio Niklitschek, Dionisia Sepulveda, Maria Cecilia Rojas, Ignacio Romero, Marcelo Baeza, Víctor CifuentesAbstract:The yeast Xanthophyllomyces Dendrorhous synthesizes the carotenoid astaxanthin, which has applications in biotechnology because of its antioxidant and pigmentation properties. However, wild-type strains produce too low amounts of carotenoids to be industrially competitive. Considering this background, it is indispensable to understand how the synthesis of astaxanthin is controlled and regulated in this yeast. In this work, the steps leading to the synthesis of the carotenoid precursor geranylgeranyl pyrophosphate (GGPP, C20) in X. Dendrorhous from isopentenyl pyrophosphate (IPP, C5) and dimethylallyl pyrophosphate (DMAPP, C5) was characterized. Two prenyl transferase encoding genes, FPS and crtE, were expressed in E. coli. The enzymatic assays using recombinant E. coli protein extracts demonstrated that FPS and crtE encode a farnesyl pyrophosphate (FPP, C15) synthase and a GGPP-synthase, respectively. X. Dendrorhous FPP-synthase produces geranyl pyrophosphate (GPP, C10) from IPP and DMAPP and FPP from IPP and GPP, while the X. Dendrorhous GGPP-synthase utilizes only FPP and IPP as substrates to produce GGPP. Additionally, the FPS and crtE genes were over-expressed in X. Dendrorhous, resulting in an increase of the total carotenoid production. Because the parental strain is diploid, the deletion of one of the alleles of these genes did not affect the total carotenoid production, but the composition was significantly altered. These results suggest that the over-expression of these genes might provoke a higher carbon flux towards carotenogenesis, most likely involving an earlier formation of a carotenogenic enzyme complex. Conversely, the lower carbon flux towards carotenogenesis in the deletion mutants might delay or lead to a partial formation of a carotenogenic enzyme complex, which could explain the accumulation of astaxanthin carotenoid precursors in these mutants. In conclusion, the FPS and the crtE genes represent good candidates to manipulate to favor carotenoid biosynthesis in X. Dendrorhous.
